Botulinum toxin neurotoxic component for treating juvenile cerebral palsy

ABSTRACT

The invention provides for the use of a presynaptic neurotoxin (for example a bacterial neurotoxin such as botulinum toxin A) for the manufacture of a medicament for the treatment of cerebral palsy in juvenile patients. The juvenile patients are preferably juveniles of up to 6 years in age.

CROSS REFERENCE

This application is a continuation of application Ser. No. 10/976,507,filed Oct. 29, 2004, which is a continuation of application Ser. No.10/155,280, filed May 22, 2002, now abandoned, which is a continuationof application Ser. No. 08/211,352, filed Jun. 27, 1994, now U.S. Pat.No. 6,395,277, which is a section 371 application from PCT/GB92/01697,filed Sep. 16, 1992, which claims priority to GB 9120306.7, filed Sep.24, 1991. The entireties of all these prior applications and patent isincorporated herein by reference.

BACKGROUND

The present invention relates to the treatment of cerebral palsy in ajuvenile patient and in particular to the promotion of normal musclegrowth in a juvenile patient suffering from dynamic contractures causedby cerebral palsy.

Cerebral palsy is a collective name given to a range of conditionscaused by brain injury caused at or around the time of birth, or in thefirst year of an infant's life. The brain injury may be caused, forexample, by trauma during delivery. It may also arise through suchcauses as trauma due to road traffic accidents or meningitis during thefirst year of life. It has been found that there is an increased risk ofcerebral palsy in prematurely born babies and, as a result of theimprovements in technology which enable premature babies to be keptalive from a much earlier age, the incidence of cerebral palsy in manycountries is actually increasing rather than falling.

Although the brain injury causing cerebral palsy is a non-progressiveinjury, its effects may change as the sufferer grows older. The largestgroup of sufferers from cerebral palsy suffer from spastic cerebralpalsy.

Spastic cerebral palsy is characterized by dynamic contractures of themuscles which impair or inhibit completely the sufferer's ability to usehis or her muscles. Moreover, muscle growth is impaired such that thelongitudinal muscles become shorter relative to their associated bonesas the infant grows older. Where the leg muscles are affected, themobility of the sufferer can be severely reduced. Conventional attemptsto cure this defect and to restore a measure of normal mobilitytypically have involved surgical intervention to alter the lengths ofthe tendons once the state has been reached at which the knee joint canno longer be straightened or the sufferer can only walk on tiptoe.

There remains a need for a treatment which allows the longitudinalmuscles to grow normally, thereby removing, or at least minimizing theneed to resort to surgical intervention. Moreover, there remains a needfor a treatment which can augment surgical intervention to improve themobility of the sufferer.

A bacterial toxin, botulinum toxin, has been used in the treatment of anumber of conditions involving muscular spasm, for example ablepharospasm, spasmodic torticollis (cervical dystonia), oromandibulardystonia and spasmodic dysphonia (laryngeal dystonia). The toxin bindsrapidly and strongly to presynaptic cholinergic nerve terminals andinhibits the exocytosis of acetylcholine by decreasing the frequency ofacetylcholine release. This results in paralysis, and hence relaxation,of the muscle afflicted by spasm.

The term Botulinum toxin is used herein as a generic term embracing thefamily of toxins produced by the anaerobic bacterium Clostridiumbotulinum and, to date, seven immunologically distinct toxins have beenidentified. These have been given the designations A, B, C, D, E, F andG. For further information concerning the properties of the variousbotulinum toxins, reference is made to the article by Jankovic & Brin,The New England Journal of Medicine, pp 1186-1194, No. 17, 1991 and tothe review by Charles L. Hatheway, Chapter 1 of the book entitled“Botulinum Neurotoxin and Tetanus Toxin” Ed. L. L. Simpson, published byAcademic Press Inc., of San Diego, Calif. 1989, the disclosures of whichare incorporated herein by reference.

The neurotoxic component of botulinum toxin has a molecular weight ofabout 150 kilodaltons and is thought to comprise a short polypeptidechain of about 50 kD which is considered to be responsible for the toxicproperties of the toxin, and a larger polypeptide chain of about 100 kDwhich is believed to be necessary to enable the toxin to penetrate thenerve. The “short” and “long” chains are linked together by means ofdisulphide bridges.

The neurotoxic polypeptide component is present in a complex withnon-toxic proteins and haemagglutinins, the molecular weight of thecomplex being in the region of 900 kD.

Botulinum toxin is obtained commercially by establishing and growingcultures of C. botulinum in a fermenter and the harvesting and purifyingthe fermented mixture in accordance with known techniques.

The “A” form of botulinum toxin is currently available commercially fromseveral sources, for example, from Porton Products Ltd., UK under thetradename “DYSPORT”, and from Allergan Inc., Irvine, Calif. under thetrade name “OCULINUM.”

It has now been found by the present inventor that children sufferingfrom cerebral palsy related dynamic muscle contractures exhibitimprovements in function following treatment with a botulinum toxin andthat such functional improvements persist when the tone reducing effectsof the toxin have worn off.

It has also been found that by administering a botulinum toxin to ajuvenile spastic mammal during its grown phase, the consequent reductionin tone of the spastic muscle enables increased longitudinal growth ofthe muscle to take place.

In a first aspect, the present invention provides a method of treating ajuvenile patient suffering from arrested muscle grown arising from thepresence of dynamic contractures of the muscle, which method comprisesadministering to the patient a therapeutically effective amount ofsubstance which blocks the release of synaptic vesicles containingacetylcholine.

The present invention also provides a method of treating a juvenilepatient suffering from cerebral palsy, which method comprisesadministering to the patient a therapeutically effective amount ofsubstance which blocks the release of synaptic vesicles containingacetylcholine.

In a further aspect the invention provides a method of treating ajuvenile patient suffering from arrested muscle grown arising from thepresence of dynamic contractures of the muscle, which method comprisesadministering to the patient a therapeutically effective amount of apresynaptic neurotoxin, for example a bacterial neurotoxin such as abotulinum toxin.

In a still further aspect the invention provides a method of eating ajuvenile patient suffering from arrested muscle growth due to a cerebralpalsy, which method comprises administering a presynaptic neurotoxin(for example a bacterial neurotoxin such as a botulinum toxin) to thepatient in a non toxic amount sufficient to reduce muscle tone andpromote improved muscle growth.

The botulinum toxin used according to the present invention preferablyis Botulinum toxin A. Botulinum toxin A is available commercially fromPorton Products Limited, UK, and from Allergan Inc., Irvine, Calif.

Administration of the toxin preferably is by means of intramuscularinjection directly into a spastic muscle, in the region of theneuromuscular junction, although alternative types of administration(e.g., sub-cutaneous injection) which can deliver the toxin directly tothe affected muscle region may be employed where appropriate. The toxincan be presented as a sterile pyrogen-free aqueous solution ordispersion and as a sterile powder for reconstitution into a sterilesolution or dispersion.

Where desired, tonicity adjusting agents such as sodium chloride,glycerol and various sugars can be added. Stabilizers such as humanserum albumin may also be included. The formulation may be preserved bymeans of a suitable pharmaceutically acceptable preservative such as aparaben, although preferably it is unpreserved.

It is preferred that the toxin is formulated in unit dosage form, forexample it can be provided as a sterile solution in a vial, or as a vialor sachet containing a lyophilised powder for reconstituting a suitablecarrier such as water for injection.

In one embodiment the toxin, e.g., botulinum toxin A is formulated in asolution containing saline and pasteurized human serum albumin, whichstabilizes the toxin. The solution is sterile filtered (0.2 micronfilter), filled into individual vials and then vacuum dried to give asterile lyophilised powder. In use, the powder can be reconstituted bythe addition of sterile unpreserved normal saline (sodium chloride 0.9%for injection).

In order for the benefits of the invention to be realized,administration of the botulinum toxin should commence before the childhas completed its growing period and fixed myostatic contracture hasoccurred. The benefits of the invention can be maximized byadministering the botulinum toxin to the child at an early stage in itsgrowing period, for example before the child reaches the age of six.

The dose of toxin administered to the patient will depend upon theseverity of the condition, e.g., the number of muscle groups requiringtreatment, the age and size of the patient and the potency of the toxin.The potency of the toxin is expressed as a multiple of the LD₅₀ value ofthe mouse, on “unit” of toxin being defined as being the equivalentamount of toxin that kills 50% of a group of mice. The definitioncurrently used in relation to the product marketed by Porton ProductsLimited. According to this definition, the potency of the botulinumtoxin A available from Porton Products Ltd. is such that one nanogramcontains 40 mouse units (units).

Typically, the dose administered to the patient will be up to about 1000units, for example, up to about 500 units, preferably in the range fromabout 80 to about 460 units per patient per treatment, although smalleror larger doses may be administered in appropriate circumstances. Thepotency of botulinum toxin, and its long duration of action means thatdoses will tend to be administered on an infrequent basis. Ultimately,however, both the quantity of toxin administered, and the frequency ofits administration will be at the discretion of the physicianresponsible for the treatment, and will be commensurate with questionsof safety and the effects produced by the toxin.

The invention will now be illustrated in greater detail by reference tothe following non-limiting examples which describe the results ofclinical studies with botulinum toxin A:

EXAMPLE 1

The Use of Botulinum Toxin A in the Management Children with CerebralPalsy

Thirty three children suffering from cerebral palsy, having a mean ageof seven years and an age range of two to seventeen years, were selectedfor participation in a clinical study.

The criteria for inclusion in the study were the presence of dynamiccontractures interfering with function, without clinical evidence offixed myostatic contracture. Before entering the study, all childrenunderwent clinical evaluation, physiotherapist's assessment and parentalassessment. All ambulatory patients underwent gait analysis usingelectrogoniometers. The children entering the study were suffering fromspastic tetraplegia, diplegia, hemiplegia or monoplegia.

The hamstrings and/or calf muscles of each patient were injected with asterile solution containing the botulinum toxin A (obtained from PortonProducts Limited, UK). Total patient doses ranged from 80 units to 460units (one unit being equivalent to the murine LD₅₀). Before injectingany muscle group, careful consideration was given to the anatomy of themuscle group, the aim being to inject the area with the highestconcentration of neuromuscular junctions. Before injecting the muscle,the position of the needle in the muscle was confirmed by putting themuscle through its range of motion and observing the resultant motion ofthe needle end. General anesthesia, local anesthesia and sedation wereused according to the age of the patient, the number of sites to beinjected and the particular needs of the patient.

Following injection, it was noted that the onset of effects was completewithin thirty-six to seventy-two hours and lasted from six to eighteenweeks. There were no systemic or local side-effects. All but one patienthad some reduction in muscle tone; the one failure occurred early in thestudy and was probably the result of the toxin dosage administered (75units) being sub-therapeutic. None of the patients developed extensivelocal hypotonicity. The majority of children had an improvement infunction both subjectively and when measured objectively with gaitanalysis.

Following injection of the calf muscle groups, an assessment was made ofthe passive dorsiflexion at the ankle. It was found that the youngerchildren displayed a marked improvement in passive dorsiflexion, butthat for children over six years there was little improvement.

This was probably due to the dynamic contracture being replaced by afixed contracture which was unresponsive to any amount of paresis.

Case Study 1

A five year old girl with moderate right hemiplegia underwent gaitanalysis and, on examination, was found to have dynamic contractures ofher calf and hamstrings. Gait analysis recordings of sagittal planmovements (with 95% confidence limits) were made prior to injection andthese revealed that throughout the gait cycle, the knee was in excessiveflexion. Gait analysis also indicated that she was in equinus throughoutthe gait cycle.

Following injection, the knee could be extended nearly to neutral duringstance and the gait analysis pattern, although still abnormal was muchimproved. The ankle traces recorded indicated that she was able todorsiflex her ankle in gait and had developed a normal range ofmovements.

Gait analysis was also undertaken at four months. At this stage theeffects of the toxin has clinically worn off and it was found that theknee flexed to the same extent in swing that it did prior to injection.However, the gain of extension in stance was largely preserved. At theankle, there was some relapse but there was still a lesser degree ofequinus.

Case Study 2

Measurements were made of the maximal extension of the knee in a groupof patients who underwent hamstring injection. Prior to injection, theyall had some degree of dynamic knee flexion contracture. Four weeksfollowing injection, this showed a highly significant improvement.

However, the one patient who was least affected developed recurvatum atthe knee following injection. After this, all patients who had a dynamicknee flexion contracture of less than fifteen degrees were excluded fromhamstring injection. Only one local side-effect from the treatment wasnoted and this was a small subcutaneous haematoma which resolved itselfin a few days.

EXAMPLE 2

The Treatment of the Hereditary Spastic Mouse with Botulinum Toxin A

In cerebral palsy there is frequently a failure of muscle growth leadingto fixed muscular contracture. This failure has also been demonstratedin the hereditary spastic mouse (Wright J and Rang M The Spastic Mouseand the search for an animal model of spasticity in human beings) Clin.Orthop. 1990, 253, 12-19.

A study has been carried out to ascertain the effect of Botulinum ToxinA on the growth of longitudinal muscle in the spastic mouse comparedwith normal siblings. Groups of spastic mice at six days old had onecalf muscle injected with either 1.2 units of Botulinum Toxin A ornormal saline.

The mice were sacrificed at maturity and the hind limbs dissected toallow measurement of the muscle and bones.

In the control group, the spastic mice has a 13% failure of longitudinalmuscle growth compared with their normal siblings. However, the musclesof the spastic mice injected with Botulinum had growth identical to thatof their normal siblings. There was no difference in growth betweennormal mice injected with saline or Botulinum.

It can be concluded that the injection of intramuscular Botulinum toxinduring the growth period of the hereditary spastic mouse allows normallongitudinal muscle growth to take place and it is believed that thisfinding may have significance in the management of cerebral palsy.

The invention has been illustrated by reference to Botulinum toxin A butit should be understood that the invention is not limited to the use ofthis toxin. For example, other Botulinum toxins may be employed.Moreover, other presynaptic neurotoxins (e.g., of bacterial origin)which act in a manner similar to botulinum toxin may also be used. Also,synthetic analogues of the botulinum toxins may be envisaged wherein the50 kD chain and/or the 100 kD chain are subjected to amino acidinsertions, deletions and/or substitutions and, provided that suchanalogues retain the general type of activity exhibited by Botulinumtoxin A, their use in the manner described hereinbefore is embraced bythe present invention. The invention is also considered to embrace theuse of substances structurally dissimilar to Botulinum toxin A, providedthat such substances possess a prolonged ability to inhibit or blockrelease of the synaptic vesicles containing acetylcholine.

1. A method for treating cerebral palsy in a juvenile patient, themethod comprising the step of administering to the patient atherapeutically effective amount of the neurotoxic component of abotulinum toxin, wherein the neurotoxic component administered to thepatient has been purified from a botulinum toxin obtained by fermentinga Clostridium botulinum, wherein the botulinum toxin is a botulinumtoxin type A.
 2. A method for treating cerebral palsy in a juvenilepatient, the method comprising the step of administering to the patienta therapeutically effective amount of the neurotoxic component of asingle botulinum toxin type, wherein the neurotoxic componentadministered to the patient has been purified from a single botulinumtoxin type obtained by fermenting a Clostridium botulinum, wherein thebotulinum toxin is a botulinum toxin type A.
 3. A method for treatingcerebral palsy in a juvenile patient, the method comprising the step ofadministering to the patient a therapeutically effective amount of theneurotoxic component of only a botulinum toxin type A, wherein theneurotoxic component administered to the patient has been purified froma botulinum toxin type A obtained by fermenting a Clostridium botulinum.4. The methods of claims 1, 2 or 3 wherein the neurotoxic component hasa molecular weight of about 150 kilodaltons.
 5. The methods of claims 1,2 or 3 wherein the botulinum toxin obtained by fermenting a Clostridiumbotulinum is a botulinum toxin complex.
 6. The methods of claims 1, 2 or3 wherein the botulinum toxin obtained by fermenting a Clostridiumbotulinum is a botulinum toxin complex with a molecular weight of about900 kilodaltons.